One of the most common diseases in both South America and Central America is Chagas disease or American Trypanosomiasis. This disease is caused by the flagellated parasite Trypanosoma cruzi and is spread by the blood-sucking insect Triatoma infestans. Once the parasite gets into the wound created by the insect bite, it spreads throughout the body and invades host cells. Inside a host cell, the parasite transforms into a non-infective amastigote, which is able to multiply very fast. When the amount of parasites inside the cell numbers about 500, the parasites transform back into the infective trypomastigote stage. Shortly after, the cell bursts, setting the parasites free in the blood from where they can infect new cells. The disease has three phases. The first phase is the acute phase, which occurs just after infection and has only mild symptoms. The second phase is the latent phase, which can have a duration of three to ten years and is asymptomatic. The third phase is the chronic phase, during which all infected tissues are deteriorating due to large-scale cell lysis, which eventually leads to the death of the patient. Seventy percent of Chagasic patients die from a heart attack caused by severe heart damage.
The molecular mechanism by which the parasite infects the host cells is very complex and has been the topic of a lot of research projects throughout the years. This research has shown that T. cruzi expresses a unique sialic acid-transferring enzyme, which is able to cleave off α-2,3 bound sialic acids and transfer them to terminal β-1,4 galactose residues: the trans-sialidase (TS). The enzyme is anchored in the parasite cell membrane by a GPI anchor, but it is also shed in the blood after cleavage by a parasite lipase enzyme. The trans-sialidase plays an essential role in the infection cycle of T. cruzi since it makes the invasion of host cells possible. Experiments have shown that when trans-sialidase activity is inhibited (for example, by using mutant cell lines that do not have sialic acid on their surface (Ciavaglia et al., 1993; Ming et al., 1993; and Schenkman et al., 1993) or by blocking the acceptor molecules on the parasite surface (Yoshida et al., 1989; Schenkman et al., 1991; and Ruiz et al., 1993) the invasion of the host cells by the parasite is inhibited. Additionally, the trans-sialidase also plays a role in the parasite's defense mechanism against the host immune system because it is used to cover the surface of the parasite with sialic acid molecules, which make it very difficult for the immune system of the host to detect the parasite.
Because the trans-sialidase enzyme is of such an important role in infection cycle and defense, the parasite developed several methods to protect the enzyme against the host immune system. First, the parasite expresses more than 200 different trans-sialidases, of which only about 15 are active (El-sayed et al., 2005). This makes it very hard for the immune system to inhibit invasion of host cells by the parasite in a normal infection cycle, especially because the parasites and their trans-sialidases reside only a short time in the bloodstream before they enter a host cell, where they are protected from the immune system. Moreover, the trans-sialidases have a very long immunodominant tail of SAPA repeats, which act as a diversion for the immune system, successfully luring away antibodies from the important catalytic site of the enzyme.
Currently, there are two drugs used to counter the disease: Benznidazole and Nifurtimox. There is very little known about their mechanism, but it is known that they induce oxidative stress in cells. Both products do not differentiate between parasites and host cells, resulting in severe side-effects for the patient. Because of these side-effects and because they have a very limited efficiency in chronic patients, these drugs have only a limited use.
Vaccination could solve these problems. A vaccine would probably be a lot more effective in treating chronic patients than the existing medication and it would have the advantage of having a preventive effect as well. GB2000968 discloses a vaccine based on killed trypanosome. However, trypomastigotes are difficult to culture in high density, and the immunogenic capacity of the vaccine is low. Several other patent applications have described a microsomal fraction (EP0003529), a glycoprotein fraction (U.S. Pat. No. 4,298,596) or a peptide (WO9316199) derived from T. cruzi as a possible vaccine. However, none of these vaccines proved to be sufficiently efficient.
Because of its essential role in the infection and due to the fact that the enzyme is both well exposed on the cell surface and present as a free molecule in the blood and, therefore, a good target for antibodies, the trans-sialidase may be a good antigen candidate for the production of a vaccine against Chagas disease. A vaccine based on the trans-sialidase enzyme is not expected to have important side-effects because there are no homologues of the trans-sialidase in humans, which means that all generated antibodies would be parasite-specific. Several authors have disclosed vaccination based on delivery of DNA encoding trans-sialidase (Costa et al., 1998; Vasconcelos et al., 2004), possibly in combination with IL-12 (Katae et al., 2002). In the study of trans-sialidase DNA vaccination of Pereira-chioccola et al. (1999), recombinant trans-sialidase was used as control. This recombinant trans-sialidase was shown to elicit trans-sialidase-inactivating antibodies and could lower the trypomastigote-induced parasitemia in mice. However, the trans-sialidase is inefficient as antigen due to the immunodominant tail. Therefore, the use of the wild-type active parasite enzyme in vaccination is not suitable, as the high doses of recombinant trans-sialidase needed could induce side-effects, especially because it was shown that active trans-sialidase is able to activate neuronal receptors (Woronowicz et al., 2004). US2005158347 discloses a multi-component vaccine against T. cruzi comprising trans-sialidase or a polynucleotide encoding trans-sialidase. However, the drawbacks related to the use of trans-sialidase, as cited above, are also valid for this multi-component vaccine.